阻断BDNF-Trk B信号通路后运动训练对脊髓损伤后大鼠痉挛状态及腰髓内GAD65表达的影响

目的:探讨阻断脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)-酪氨酸激酶受体 B(tropomyosin-related kinase B,TrkB)信号通路后运动训练对脊髓损伤(spinal cord injury,SCI)大鼠后肢痉挛以及腰髓内谷氨酸脱羧酶65(glutamic acid decarboxylase 65,GAD65)表达的影响。方法:将60只雌性SD大鼠随机分为假手术组(Sham组)、损伤+磷酸盐缓冲液组(Sed-PBS组)、运动+磷酸盐缓冲液组(TT-PBS组)、损伤+TrkB 阻滞剂组(Sed-TrkB/Fc组)以及运动+TrkB 阻滞剂组(TT-TrkB/Fc组)。采用改良Allen撞击法建立T10不完全脊髓损伤后痉挛模型,在SCI后第7天,于Sed-TrkB/Fc组和 TT-TrkB/Fc组植入灌有TrkB 阻滞剂(TrkB/Fc)的Alzet渗透压泵,其余三组渗透压泵中则注入0.01M 磷酸盐缓冲液(phosphate-buffered saline,PBS)进行对照。SCI后第8天,TT-PBS组和TT-TrkB/Fc组进行减重平板训练(treadmill training),每周使用H反射的Hmax/Mmax比值对术后大鼠后肢的痉挛状态进行评估,于术后5周采用Western Blot和免疫组化技术检测各组大鼠损伤远段脊髓GAD65的表达情况,并对大鼠小腿三头肌Hmax/Mmax比值与脊髓前角GAD65相对表达量之间的相关性进行分析。结果:术后,除假手术组外,其余4组大鼠后肢痉挛程度均逐渐加重。从第3周开始,与Sed-PBS组、Sed-TrkB/Fc 组和TT-TrkB/Fc组相比,TT-PBS组Hmax/Mmax比值明显降低(P<0.05);在术后第4—5周,TT-TrkB/Fc组比值也有下降(P<0.05)。5组大鼠GAD65免疫组化和Western Blot结果显示,各组GAD65的相对蛋白含量均低于假手术组(P<0.05),但TT-PBS 组明显多于除Sham组外的其他3组(P<0.05);TT-TrkB/Fc 组GAD65的相对蛋白表达量与Sed-PBS 组和 Sed-TrkB/Fc 组相比也有所增加(P<0.05)。相关性分析显示,SCI后第35天,大鼠小腿三头肌Hmax/Mmax比值与脊髓前角GAD65相对表达量之间呈负相关(P<0.001)。结论:运动训练可以改善SCI后的痉挛状态,而阻断BDNF-TrkB信号通路可抑制运动训练对不完全性SCI大鼠后肢痉挛的缓解和损伤远段脊髓GAD65表达的促进作用,且SCI后的痉挛状态的改善可能与脊髓前角GAD65相对表达量相关。

Effects of exercise training on the expression of GAD65 after blocking BDNF-TrkB pathway in spastic rats with spinal cord injury

Objective: To investigate the effects of treadmill training on the spasticity and the expression of glutamic acid decarboxylase 65(GAD65)after blocking of Brain-Derived Neurotrophic Factor (BDNF)- Tyrosine kinase receptor B (TrkB) signaling pathway in rats with incomplete spinal cord injury (SCI). Method: 60 female SD rats were randomly divided into 5 groups: sham operation group, injury+phosphate-buffered saline group (Sed-PBS group), Treadmill Training+phosphate-buffered saline group (TT-PBS group), injury+TrkB/Fc group (Sed-TrkB/Fc group) and Treadmill Training+TrkB/Fc group (TT-TrkB/Fc group). The spastic rat model with T10 incomplete SCI was induced by modified Allen's method. In the 7th day after SCI, the Alzet osmotic pump with TrkB blocker(TrkB/Fc) was implanted into rats of Sed-TrkB/Fc group and TT-TrkB/Fc group, while the Alzet osmotic pump of the three other groups were filled with 0.01M PBS. 8 days after SCI, the TT-PBS group and TT-TrkB/Fc group were underwent body-weight support treadmill training. Then we evaluated the spasticity of rats after the operation through the Hmax/Mmax ratio every week. The expression of GAD65 in injured spinal cord was examined by Western Blot and immunohistochemistry at the 5th week post injury. The correlation between the Hmax/Mmax ratio of triceps crus and the relative expression of GAD65 in the anterior horn of spinal cord in rats was also analyzed. Result: After the operation, spasticity gradually aggravated in other 4 groups except the sham operation group. At the third week after SCI, the ratio of Hmax/Mmax in the TT-PBS group was significantly lower than that of the other 3 groups (P<0.05), and at the 4-5th week, it was also lower in TT-TrkB/Fc group (P<0.05). The immunohistochemistry and the Western blot results showed that, compared with the sham operation group, the relative protein expression of GAD65 in other 4 groups were decreased (P<0.05). While the TT-PBS group was significantly higher than that of the other 3 groups (P<0.05), and the TT-TrkB/Fc group was also higher than that of the Sed-PBS group and Sed-TrkB/Fc group (P<0.05). The correlation analysis showed that in the 35th day after 5d, there was a negative correlation between the Hmax/Mmax ratio of triceps crus and the relative expression of GAD65 in the anterior horn of spinal cord in rats(P<0.001). Conclusion: Exercise training can alleviate spasticity after SCI. Blocking the BDNF-TrkB signaling pathway can inhibit the effect of exercise training on the relief of spasticity and the expression of GAD65 in lumbar spinal cord of rats with incomplete SCI. And the relief of spasticity after SCI may be related to the relative expression of GAD65 in the anterior horn of spinal cord.